Cationic Polymer Based Wired Enzyme Formulations for Use in Analyte Sensors

ABSTRACT

Embodiments of the invention include analyte-responsive compositions and electrochemical analyte sensors having a sensing layer that includes an analyte-responsive enzyme and a cationic polymer. Also provided are systems and methods of making the sensors and using the electrochemical analyte sensors in analyte monitoring.

BACKGROUND OF THE INVENTION

Enzyme-based biosensors are devices in which ananalyte-concentration-dependent biochemical reaction signal is convertedinto a measurable physical signal, such as an optical or electricalsignal. Such biosensors are widely used in the detection of analytes inclinical, environmental, agricultural and biotechnological applications.Analytes that can be measured in clinical assays of fluids of the humanbody include, for example, glucose, lactate, cholesterol, bilirubin andamino acids. The detection of analytes in biological fluids, such asblood, is important in the diagnosis and the monitoring of manydiseases.

Biosensors that detect analytes via electrical signals, such as current(amperometric biosensors) or charge (coulometric biosensors), are ofspecial interest because electron transfer is involved in thebiochemical reactions of many important bioanalytes. Systems includethose intended for in vitro use (e.g., test strips) and those intendedfor in vivo use (e.g., in which at least a portion of a sensor ispositioned in a user).

Typically, systems employ at least one working electrode and a sensinglayer that includes an analyte-responsive enzyme in proximity thereto.Analyte monitoring systems may vary depending on a variety of factorssuch as the particular technology (e.g., amperometric, coulometry,optical, etc.) and the sensing material. For example, the sensing layerwill vary depending on the analyte(s) of interest such as glucoseoxidase or glucose dehydrogenase when the analyte of interest isglucose, and may employ a mediator and/or other components.

As analyte monitoring devices and methods, particularly glucosemonitoring, becomes increasingly important for disease control, there isa continued interest for development of new analyte monitoring systems,including new sensing layers, that are highly stable, and that areversatile in that they may be employed with a variety of differentenzymes. Such sensing layers that simplify manufacturing processes arealso desirable.

SUMMARY OF THE INVENTION

Embodiments of the invention include electrochemical analyte sensorshaving a sensing layer that includes an analyte-responsive enzyme and acationic polymer, where the sensing layer is positioned proximate to aworking electrode of the sensor. A mediator, such as one that includes atransition metal complex, may be employed. In certain embodiments, themediator is non-covalently associated, i.e., not physically attached to,the cationic polymer. Also provided are systems and methods of makingand using the electrochemical analyte sensors.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 shows a block diagram of an embodiment of a data monitoring andmanagement system according to embodiments of the invention;

FIG. 2 shows a block diagram of an embodiment of the transmitter unit ofthe data monitoring and management system of FIG. 1;

FIG. 3 shows a block diagram of an embodiment of the receiver/monitorunit of the data monitoring and management system of FIG. 1;

FIG. 4 shows a schematic diagram of an embodiment of an analyte sensoraccording to the embodiments of the invention;

FIGS. 5A-5B show a perspective view and a cross sectional view,respectively of another embodiment an analyte sensor;

FIG. 6 shows a comparison of the linearity of the cationic polymer basedGOx sensing layer (triangle) versus the redox polymer and cross-linkerbased sensing layer (diamond);

FIG. 7 shows a comparison of the response time of the cationic polymerbased GOx sensing layer (cross hatched) versus the redox polymer andcross-linker based sensing layer (no fill);

FIG. 8 shows a comparison of the stability at 37° C. of the cationicpolymer based GOx sensing layer (solid lines) versus the redox polymerand cross-linker based sensing layer (dashed lines);

FIG. 9 shows a comparison of the stability at 65° C. of the cationicpolymer based GOx sensing layer (solid lines) versus the redox polymerand cross-linker based sensing layer (dashed lines);

FIG. 10 shows a comparison of the linearity of the cationic polymerbased FADGDH sensing layer (square) versus the redox polymer andcross-linker based sensing layer (diamond);

FIG. 11 shows a comparison of the response time of the cationic polymerbased FADGDH sensing layer versus the redox polymer and cross-linkerbased sensing layer; and

FIG. 12 shows a comparison of the stability at 37° C. of the cationicpolymer based FADGDH sensing layer versus the redox polymer andcross-linker based sensing layer;

The figures shown herein are not necessarily drawn to scale, with somecomponents and features being exaggerated for clarity.

DETAILED DESCRIPTION OF THE INVENTION

Before the embodiments of the invention is described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the embodiments of the invention will be limited only bythe appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the embodiments of the invention, somepotential and exemplary methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the embodimentsof the disclosure supercedes any disclosure of an incorporatedpublication to the extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the application. Nothing hereinis to be construed as an admission that the embodiments of the inventionare not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be independentlyconfirmed.

Sensing Layer Enzyme Formulations

Sensing layers of analyte monitoring systems, e.g., glucose monitoringsystems, have been described for in vitro and in vivo systems. WiredEnzyme™ systems have been described, e.g., in U.S. Pat. Nos. 5,262,035;5,543,326; XXXXX, the disclosures of which are incorporated by reference(See also “A Continuous Glucose Sensor Based on Wired Enzyme™Technology—Results from a 3-Day Trial in Patients with Type 1 Diabetes”,Feldman et al., Diabetes Technol Ther. 2003; 5(5):769-79). For example,Wired Enzyme™ sensing layers have included an analyte responsive enzyme,redox polymer and crosslinker so that electrons are effectivelytransferred from the analyte of interest (e.g., glucose) to a workingelectrode of the sensor by way of the Wired Enzyme™ complex, i.e., theenzymes are electrically wired.

Embodiments of the invention include electrochemical analytecompositions and sensors having a sensing layer that includes ananalyte-responsive enzyme, a cationic polymer, and a redox mediator. Incertain embodiments, the redox mediator is covalently associated withthe cationic polymer, such as by a covalent bond tethering the redoxmediator to the cationic polymer. In other embodiments, the redoxmediator is non-covalently associated with the cationic polymer, such asby ionic interactions, hydrophobic interactions, hydrogen bonds, Van derWaals forces, i.e. “London dispersion forces”, Dipole-dipole bonds, aswell as physical interactions between the molcues that results instabalizing and immobilizng the mediator on the sensor. In still otherembodiments, the mediator is not diffusibly assocaited with the cationicpolymer and the analyte-responsive enzyme. Advantageously, such thesensing layers do not require a cross linker. A mediator may thus befreely moveable, i.e., a diffusible or non-immobilized mediator.

Mediators that may be used include those that include transition metalcomplexes such as osmium and ruthenium. Also provided are systems andmethods of using the electrochemical analyte sensors in analytemonitoring. The sensing layers described herein provide high stability,especially when compared to systems that do not include Wired Enzyme™systems, and may be used with a variety of different enzymes. Inaddition, the sensing layers have a lower manufacturing cost, e.g., byavoiding multiple synthesis steps as compared to conventional sensinglayers used in electrochemical analyte sensors.

Any ratio of cationic polymer to mediator to enzyme can be used thatprovides the optimal desirable properties, including, but limited to,linearity in sensitivity to an analyte over a range of concentrations,stability at various analyte concentrations and at various temperatures,and the like. As will be appreciated by one of skill in the art, theratio will differ based on the specific cationic polymer, optional redoxmediator mediator, and analyte-responsive enzyme used in theformulation. By way of example, the cationic polymer to mediator andenzyme by weight may range from about 1:20 to about 32:1, and a mediatorand enzyme ratio may range from about 1:10 to about 20:1. For example,this range is from about 1:20 to about 25:1, including about 1:15 toabout 15:1, about 1:10 to about 10:1, about 1:4 to about 1:10, etc. Incertain embodiments, the ratio of cationic polymer to mediator to enzymecan be about 1:1:2.

The sensing layer enzyme formulation may be used with a variety ofbiosensors. Examples of such biosensors include, but are not limited to,glucose sensors and lactate sensors. (See, for example, U.S. Pat. Nos.6,175,752 and 6,134,461). The coating process may comprise any commonlyused technique, such as spin-coating, dip-coating, or dispensingdroplets of the sensing layer solution, and the like, followed by curingunder ambient conditions, e.g., for about 1 to 2 days. The particulardetails of the coating process (such as dip duration, dip frequency,number of dips, or the like) may vary depending on the nature (i.e.,viscosity, concentration, composition, or the like) of the cationicpolymer, the analyte-responsive enzyme, and any other optionalcomponents such as solvent, buffer, etc., for example. Conventionalequipment may be used for the coating process, such as a DSG DlL-160dip-coating or casting system of NTMA Technology in the United Kingdom.

Elements of the sensing layer enzyme formulation are described ingreater detail below.

Analyte-Responsive Enzyme

Any analyte-responsive enzyme capable of catalyzing the electrooxidationor electroreduction of a biomolecule is suitable for use in the sensinglayer enzyme formulation. Exemplary analyte-responsive enzymes includeanalyte-responsive oxidases and analyte-responsive dehydrogenases. Ingeneral, the selection of the analyte-responsive enzyme will depend onthe analyte to be detected. For example, a glucose oxidase (GOx) orglucose dehydrogenase (GDH), such as, for example, pyrroloquinolinequinone glucose dehydrogenase (PQQGDH), or flavin adenine dinucleotideglucose dehydrogenase (FADGDH) may be used when the analyte is glucose.A lactate oxidase may fill this role when the analyte is lactate. Otherenzymes can be used for other analytes. These enzymes catalyze theelectrolysis of an analyte by transferring electrons between the analyteand the electrode via the redox mediator.

In certain embodiments, the composition will include ananalyte-responsive glucose oxidase. In other embodiments, thecomposition will include an analyte-responsive dehydrogenase. Adehydrogenase is an enzyme that oxidizes a substrate by transferring oneor more protons and a pair of electrons to an acceptor. Generally, anydehydrogenase may be used in embodiments of the invention, including,for example, glucose dehydrogenase (GDH). Examples of dehydrogenasesinclude, but are not limited to, aldehyde dehydrogenase, acetaldehydedehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase, lactatedehydrogenase, pyruvate dehydrogenase, glucose-6-phosphatedehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, isocitratedehydrogenase, alpha-ketoglutarate dehydrogenase, succinatedehydrogenase, and malate dehydrogenase.

In some embodiments, where the analyte-responsive dehydrogenase, suchas, for example, glucose dehydrogenase, the analyte-responsivedehydrogenase may further be complexed with a co-factor that providesfor electron transfer to a redox mediator. Suitable co-factors include,but are not limited to, Flavin adenine dinucleotide (FAD), nicotinamideadenine dinucleotide (NAD), pyrroloquinoline quinone (PQQ), and the likethat provide for electron transfer to a redox mediator.

Cationic Polymer

Cationic polymers suitable for use in the sensing layer formulations maybe any linear or branched cationic polymer that provides the desirableproperties and is compatible for use with the analyte-responsive enzymeand the optional redox mediator and transition metal complex. Exemplarycationic polymers include, but are not limited to, a polyallylamine(PAH); a polyethyleneimine (PEI); a poly(L-lysine) (PLL); apoly(L-arginine) (PLA); a polyvinylamine homo- or copolymer; apoly(vinylbenzyl-tri-C₁-C₄-alkylammonium salt); a polymer of analiphatic or araliphatic dihalide and an aliphaticN,N,N′,N′-tetra-C₁-C₄-alkyl-alkylenediamine; a poly(vinylpyridin) orpoly(vinylpyridinium salt); a poly(N,N-diallyl-N,N-di-C₁-C₄-alkyl-ammoniumhalide); a homo- or copolymer ofa quaternized di-C₁-C₄-alkyl-aminoethyl acrylate or methacrylate;POLYQUAD™; a polyaminoamide; and the like.

Exemplary cationic polymers include copolymer of hydroxyethyl celluloseand diallyldimethylammonium chloride, the copolymer of acrylamide anddiallyldimethylammonium chloride, the copolymer of vinyl pyrrolidone anddimethylamino ethylmethacrylate methosulfate, the copolymer ofacrylamide and betamethacrylyloxyethyl trimethyl ammonium chloride, thecopolymer of polyvinyl pyrrolidone and imidazolimine methochloride, thecopolymer of diallyldimethyl ammonium chloride and acrylic acid, thecopolymer of vinyl pyrrolidone and methacrylamidopropyl trimethylammonium chloride, the methosulfate of the copolymer ofmethacryloyloxyethyl trimethylammonium and methacryloyloxyethyldimethylacetylammonium, quaternized hydroxyethyl cellulose;dimethylsiloxane3-(3-((3-cocoamidopropyl)dimethylammonio)-2-hydroxyprpoxy)propylgroupterminated acetate; the copolymer of aminoethylaminopropylsiloxaneand dimethylsiloxan; the polyethylene glycol derivative ofaminoethylaminopropylsiloxane/dimethylsiloxan-copolymer and cationicsilicone polymers; and the like.

Other exemplary cationic polymers include cationic modified proteinderivatives or cationic modified protein hydrolysates and are, forexample, known under the INCI designations Lauryldimonium HydroxypropylHydrolyzed Wheat Protein, Lauryldimonium Hydroxypropyl HydrolyzedCasein, Lauryldimonium Hydroxypropyl Hydrolyzed Collagen, LauryldimoniumHydroxypropyl Hydrolyzed Keratin, Lauryldimonium HydroxypropylHydrolyzed Silk, Lauryldimonium Hydroxypropyl Hydrolyzed Soy Protein, orHydroxypropyltrimonium Hydrolyzed Wheat, HydroxypropyltrimoniumHydrolyzed Casein, Hydroxypropyltrimonium Hydrolyzed Collagen,Hydroxypropyltrimonium Hydrolyzed Keratin, HydroxypropyltrimoniumHydrolyzed Rice Bran Protein, Hydroxypropyltrimonium Hydrolyzed Silk,Hydroxypropyltrimonium Hydrolyzed Soy Protein, HydroxypropyltrimoniumHydrolyzed Vegetable Protein; and the like.

Exemplary cationically derived protein hydrolysates are substancemixtures, which, for example, receive glycidyl trialkyl ammonium saltsor 3-halo-2-hydroxypropyl trialkyl ammonium salts via the conversion ofalkaline, acidic, or enzyme-hydrolyzed proteins. Proteins that are usedas starting materials for the protein hydrolysates can be of plant oranimal origin. Starting materials may include, for example, keratin,collagen, elastin, soy protein, rice protein, milk protein, wheatprotein, silk protein, almond protein, and the like. The hydrolysis mayresult in material mixtures with mole masses in the range of about 100to about 50,000. Some mean mole masses may be in the range of about 500to about 1,000. It is advantageous if the cationically derived proteinhydrolysates have one or two long C8 to C22 alkyl chains and two or oneshort C1 to C4 alkyl chain accordingly.

Other exemplary cationic polymers include cationic silicon polymers.Cationic silicon polymers either have at least one least one ammoniumgroup, examples are POLYSILICONE-9; inclduing diquaternary polysiloxanesand those having the chemical name dimethylsiloxane,3-(3-((3-cocoamidopropyl)dimethylammonio)-2-hydroxyprpoxy)propyl groupterminated acetate (CAS 134737-05-6), defined in the CTFA asQUATERNIUM-80 and sold under the trade names Abil Quat™ 3270, Abil™ Quat3272, and Abil™ Quat 3474 by the company Th. Goldschmidt AG, Germany;another exemplary cationic silicon polymer isaminoethylaminopropylsiloxane/dimethylsiloxan-copolymer emulsion, sold,e.g., as GE Toshiba Silicone™ and Dow Corning 2-8566™ (CTFA:AMODIMETHICONE), another exemplary cationic silicon polymer is thepolyethylene glycol derivative ofaminoethylaminopropylsiloxane/dimethylsiloxan-copolymer (CTFA: PEG-7AMODIMETHICONE). Other exemplary silicone polymers include (as definedin CTFA 10^(th) Edition): SILICONE QUATERNIUM-1, SILICONE QUATERNIUM-2,SILICONE QUATERNIUM-2 PANTHENOL, SILICONE QUATERNIUM-3, SILICONEQUATERNIUM-4, SILICONE QUATERNIUM-5, SILICONE QUATERNIUM-6, SILICONEQUATERNIUM-7, SILICONE QUATERNIUM-8, SILICONE QUATERNIUM-9, SILICONEQUATERNIUM-10, SILICONE QUATERNIUM-11 and SILICONE QUATERNIUM-12.

Redox Mediator Comprising a Transition Metal Complex

In certain embodiments, the sensing layers may include a mediator. Anysuitable mediator may be employed. Embodiments include mediators thatare diffusing, non-immobilized mediators in that they are not bound tothe cationic polymer (or enzyme). Mediators include ferricyanide,phenanthroline quinine, such as 1,10-Phenanthroline quinone (see U.S.Pat. No. 6,736,957, incorporated herein by reference), ferrocene, andtransition metal complexes. For example, compounds having the formula 1are examples of transition metal complexes of the embodiments of theinvention:

M is a transition metal and is typically iron, cobalt, ruthenium,osmium, or vanadium. Ruthenium and osmium are particularly suitable forredox mediators.

L is a bidentate ligand containing at least one imidazole ring. Oneexample of L is a 2,2′-biimidazole having the following structure 2:

R₁ and R₂ are substituents attached to two of the 2,2′-biimidazolenitrogens and are independently substituted or unsubstituted alkyl,alkenyl, or aryl groups. Generally, R₁ and R₂ are unsubstituted C1 toC12 alkyls. Typically, R₁ and R₂ are unsubstituted C1 to C4 alkyls. Insome embodiments, both R₁ and R₂ are methyl.

R₃, R₄, R₅, and R₆ are substituents attached to carbon atoms of the2,2′-biimidazole and are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,—OH, alkoxy, —NH₂₅ alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino,alkylthio, alkenyl, aryl, or alkyl. Alternatively, R₃ and R₄ incombination or R₅ and R₆ in combination independently form a saturatedor unsaturated 5- or 6-membered ring. An example of this is a2,2′-bibenzoimidazole derivative. Typically, the alkyl and alkoxyportions are C1 to C12. The alkyl or aryl portions of any of thesubstituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. Generally, R₃, R₄, R₅, and R₆ areindependently —H or unsubstituted alkyl groups. Typically, R₃, R₄, R₅,and R₆ are —H or unsubstituted C1 to C12 alkyls. In some embodiments,R₃, R₄, R₅, and R₆ are all —H.

Another example of L is a 2-(2-pyridyl)imidazole having the followingstructure 3:

R′₁ is a substituted or unsubstituted aryl, alkenyl, or alkyl.Generally, R′₁ is a substituted or unsubstituted C1-C12 alkyl. R′₁ istypically methyl or a C1-C12 alkyl that is optionally substituted with areactive group.

R′₃, R′₄, R_(a), R_(b), R_(c) and R_(d) are independently selected fromH, F, Cl, Br, I, NO₂, CN, CO₂H, SO₃H, NHNH₂, SH, alkoxylcarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R_(c) and R_(d) in combination or R′₃ and R′₄ incombination can form a saturated or unsaturated 5- or 6-membered ring.Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl oraryl portions of any of the substituents are optionally substituted by—F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except onaryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally,R′₃, R′₄, R_(a), R_(b), R_(c) and R_(d) are independently —H orunsubstituted alkyl groups. Typically, R_(a) and R_(c) are —H and R′₃,R′₄, R_(b), and R_(d) are —H or methyl.

c is an integer indicating the charge of the complex. Generally, c is aninteger selected from −1 to −5 or +1 to +5 indicating a positive ornegative charge. For a number of osmium complexes, c is +2 or +3.

X represents counter ion(s). Examples of suitable counter ions includeanions, such as halide (e.g., fluoride, chloride, bromide or iodide),sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, andcations (preferably, monovalent cations), such as lithium, sodium,potassium, tetralkylammonium, and ammonium. Preferably, X is a halide,such as chloride. The counter ions represented by X are not necessarilyall the same.

d represents the number of counter ions and is typically from 1 to 5.

The term “alkyl” includes linear or branched, saturated aliphatichydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, tert-butyl and the like. Unless otherwise noted, theterm “alkyl” includes both alkyl and cycloalkyl groups.

The term “alkoxy” describes an alkyl group joined to the remainder ofthe structure by an oxygen atom. Examples of alkoxy groups includemethoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, and thelike. In addition, unless otherwise noted, the term ‘alkoxy’ includesboth alkoxy and cycloalkoxy groups.

The term “alkenyl” describes an unsaturated, linear or branchedaliphatic hydrocarbon having at least one carbon-carbon double bond.Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl,1-butenyl, 2-methyl-1-propenyl, and the like.

A “reactive group” is a functional group of a molecule that is capableof reacting with another compound to couple at least a portion of thatother compound to the molecule. Reactive groups include carboxy,activated ester, sulfonyl halide, sulfonate ester, isocyanate,isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine,acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine,alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate,halotriazine, imido ester, maleimide, hydrazide, hydroxy, andphoto-reactive azido aryl groups. Activated esters, as understood in theart, generally include esters of succinimidyl, benzotriazolyl, or arylsubstituted by electron-withdrawing groups such as sulfo, nitro, cyano,or halo groups; or carboxylic acids activated by carbodiimides.

A “substituted” functional group (e.g., substituted alkyl, alkenyl, oralkoxy group) includes at least one substituent selected from thefollowing: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, —NH₂, alkylamino,dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido,hydrazino, alkylthio, alkenyl, and reactive groups.

L₁, L₂, L₃ and L₄ are ligands attached to the transition metal via acoordinative bond. L₁, L₂, L₃ and L₄ can be monodentate ligands or, inany combination, bi-, ter-, or tetradentate ligands For example, L₁, L₂,L₃ and L₄ can combine to form two bidentate ligands such as, forexample, two ligands selected from the group of substituted andunsubstituted 2,2′-biimidazoles, 2-(2-pyridyl)imidizoles, and2,2′-bipyridines

Examples of other L₁, L₂, L₃ and L₄ combinations of the transition metalcomplex include:

-   (A) L₁ is a monodentate ligand and L₂, L₃ and L₄ in combination form    a terdentate ligand;-   (B) L₁ and L₂ in combination are a bidentate ligand, and L₃ and L₄    are the same or different monodentate ligands;-   (C) L₁ and L₂ in combination, and L₃ and L₄ in combination form two    independent bidentate ligands which can be the same or different;    and-   (D) L₁, L₂, L₃ and L₄ in combination form a tetradentate ligand.

Examples of suitable monodentate ligands include, but are not limitedto, —F, —Cl, —Br, —I, —CN, —SCN, —OH, H₂O, NH₃, alkylamine,dialkylamine, trialkylamine, alkoxy or heterocyclic compounds. The alkylor aryl portions of any of the ligands are optionally substituted by —F,—Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group. Any alkylportions of the monodentate ligands generally contain 1 to 12 carbons.More typically, the alkyl portions contain 1 to 6 carbons. In otherembodiments, the monodentate ligands are heterocyclic compoundscontaining at least one nitrogen, oxygen, or sulfur atom. Examples ofsuitable heterocyclic monodentate ligands include imidazole, pyrazole,oxazole, thiazole, pyridine, pyrazine and derivatives thereof. Suitableheterocyclic monodentate ligands include substituted and unsubstitutedimidazole and substituted and unsubstituted pyridine having thefollowing general formulas 4 and 5, respectively:

With regard to formula 4, R₇ is generally a substituted or unsubstitutedalkyl, alkenyl, or aryl group. Typically, R₇ is a substituted orunsubstituted C1 to C12 alkyl or alkenyl. The substitution of innercoordination sphere chloride anions by imidazoles does not typicallycause a large shift in the redox potential in the oxidizing direction,differing in this respect from substitution by pyridines, whichtypically results in a large shift in the redox potential in theoxidizing direction.

R₈, R₉ and R₁₀ are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H,—SO₃H, —NHNH₂, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino, dialkylamino,alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R₉ and R₁₀, in combination, form a fused 5 or 6-memberedring that is saturated or unsaturated. The alkyl portions of thesubstituents generally contain 1 to 12 carbons and typically contain 1to 6 carbon atoms. The alkyl or aryl portions of any of the substituentsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. In some embodiments, R₈, R₉ andR₁₀ are —H or substituted or unsubstituted alkyl. Preferably, R₈, R₉ andR₁₀ are —H.

With regard to Formula 5, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are independently—H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Thealkyl or aryl portions of any of the substituents are optionallysubstituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except for aryl portions), alkoxy, alkylthio, aryl, ora reactive group. Generally, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are —H, methyl,C1-C2 alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 loweralkyl substituted with a reactive group.

One example includes R₁₁ and R₁₅ as —H, R₁₂ and R₁₄ as the same and —Hor methyl, and R₁₃ as —H, C1 to C12 alkoxy, —NH₂, C1 to C12 alkylamino,C2 to C24 dialkylamino, hydrazino, C1 to C12 alkylhydrazino,hydroxylamino, C1 to C12 alkoxyamino, C1 to C12 alkylthio, or C1 to C12alkyl. The alkyl or aryl portions of any of the substituents areoptionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, ora reactive group.

Examples of suitable bidentate ligands include, but are not limited to,amino acids, oxalic acid, acetylacetone, diaminoalkanes,ortho-diaminoarenes, 2,2′-biimidazole, 2,2′-bioxazole, 2,2′-bithiazole,2-(2-pyridyl)imidazole, and 2,2′-bipyridine and derivatives thereof.Particularly suitable bidentate ligands for redox mediators includesubstituted and unsubstituted 2,2′-biimidazole, 2-(2-pyridyl)imidazoleand 2,2′-bipyridine. The substituted 2,2′ biimidazole and2-(2-pyridyl)imidazole ligands can have the same substitution patternsdescribed above for the other 2,2′-biimidazole and2-(2-pyridyl)imidazole ligand. A 2,2′-bipyridine ligand has thefollowing general formula 6:

R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are independently —H, —F, —Cl,—Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂₅ alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, or alkyl. Typically,the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portionsof any of the substituents are optionally substituted by —F, —Cl, —Br,—I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group.

Specific examples of suitable combinations of R₁₆, R₁₇, R₁₈, R₁₉, R₂₀,R₂₁, R₂₂ and R₂₃ include R₁₆ and R₂₃ as H or methyl; R₁₇ and R₂₂ as thesame and —H or methyl; and R₁₉ and R₂₀ as the same and —H or methyl. Analternative combination is where one or more adjacent pairs ofsubstituents R₁₆ and R₁₇, on the one hand, and R₂₂ and R₂₃, on the otherhand, independently form a saturated or unsaturated 5- or 6-memberedring. Another combination includes R₁₉ and R₂₀ forming a saturated orunsaturated five or six membered ring.

Another combination includes R₁₆, R₁₇, R₁₉, R₂₀, R₂₂ and R₂₃ as the sameand —H and R₁₈ and R₂₁ as independently —H, alkoxy, —NH₂, alkylamino,dialkylamino, alkylthio, alkenyl, or alkyl. The alkyl or aryl portionsof any of the substituents are optionally substituted by —F, —Cl, —Br,—I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group. As an example,R₁₈ and R₂₁ can be the same or different and are —H, C1-C6 alkyl, C1-C6amino, C1 to C12 alkylamino, C2 to C12 dialkylamino, C1 to C12alkylthio, or C1 to C12 alkoxy, the alkyl portions of any of thesubstituents are optionally substituted by a —F, —Cl, —Br, —I, aryl, C2to C12 dialkylamino, C3 to C18 trialkylammonium, C1 to C6 alkoxy, C1 toC6 alkylthio or a reactive group.

Examples of suitable terdentate ligands include, but are not limited to,diethylenetriamine, 2,2′,2″-terpyridine, 2,6-bis(N-pyrazolyl)pyridine,and derivatives of these compounds. 2,2′,2″-terpyridine and2,6-bis(N-pyrazolyl)pyridine have the following general formulas 7 and 8respectively:

With regard to formula 7, R₂₄, R₂₅ and R₂₆ are independently —H orsubstituted or unsubstituted C1 to C12 alkyl. Typically, R₂₄, R₂₅ andR₂₆ are —H or methyl and, in some embodiments, R₂₄ and R₂₆ are the sameand are —H. Other substituents at these or other positions of thecompounds of formulas 7 and 8 can be added.

With regard to formula 8, R₂₇, R₂₈ and R₂₉ are independently —H, —F,—Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy, —NH₂₅ alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl. Thealkyl or aryl portions of any of the substituents are optionallysubstituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, ora reactive group. Typically, the alkyl and alkoxy groups are C1 to C12and, in some embodiments, R₂₇ and R₂₉ are the same and are —H.

Examples of suitable tetradentate ligands include, but are not limitedto, triethylenetriamine, ethylenediaminediacetic acid, tetraazamacrocycles and similar compounds as well as derivatives thereof.

Examples of suitable transition metal complexes are illustrated usingFormula 9 and 10:

With regard to transition metal complexes of formula 9, the metal osmiumis complexed to two substituted 2,2′-biimidazole ligands and onesubstituted or unsubstituted 2,2′-bipyridine ligand. R₁, R₂, R₃, R₄, R₅,R₆, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, c, d, and X are the same asdescribed above.

In one embodiment, R₁ and R₂ are methyl; R₃, R₄, R₅, R₆, R₁₆, R₁₇, R₁₉,R₂₀, R₂₂ and R₂₃ are —H; and R₁₈ and R₂₁ are the same and are —H,methyl, or methoxy. Preferably, R₁₈ and R₂₁ are methyl or methoxy.

In another embodiment, R₁ and R₂ are methyl; R₃, R₄, R₅, R₆, R₁₆, R₁₇,R₁₈, R₁₉, R₂₀, R₂₂ and R₂₃ are —H; and R₂₁ is halo, C1 to C12 alkoxy, C1to C12 alkylamino, or C2 to C24 dialkylamino. The alkyl or aryl portionsof any of the substituents are optionally substituted by —F, —Cl, —Br,—I, alkylamino, dialkylamino, trialkylammonium (except on arylportions), alkoxy, alkylthio, aryl, or a reactive group. For example,R₂₁ is a C1 to C12 alkylamino or C2 to C24 dialkylamino, the alkylportion(s) of which are substituted with a reactive group, such as acarboxylic acid, activated ester, or amine. Typically, the alkylaminogroup has 1 to 6 carbon atoms and the dialkylamino group has 2 to 8carbon atoms.

With regard to transition metal complexes of formula 10, the metalosmium is complexed to two substituted 2,2′-biimidazole ligands and onesubstituted or unsubstituted 2-(2-pyridyl)imidazole ligand. R₁, R₂, R₃,R₄, R₅, R₆, R′₁, R′₃, R′₄, R_(a), R_(b), R_(c), R_(d), c, d, and X arethe same as described above.

In one embodiment, R₁ and R₂ are methyl; R₃, R₄, R₅, R₆, R′₃, R′₄ andR_(d) are independently —H or methyl; R_(a) and R_(c) are the same andare —H; and R_(b) is C1 to C12 alkoxy, C1 to C12 alkylamino, or C2 toC24 dialkylamino. The alkyl or aryl portions of any of the substituentsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group.

A list of specific examples of transition metal complexes withrespective redox potentials is shown in Table A.

TABLE A Redox Potentials of Selected Transition Metal Complexes ComplexStructure E_(1/2)(vs Ag/AgCl)/mV* I

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-dimethylamino-2,2′-bipyridine)]Cl₃ −110 II

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-methylamino-2,2′-bipyridine)]Cl₃ −100 III

  [OS(1,1′-dimethyl-2,2′-biimidazole)₂(4-bromo- 2,2′-bipyridine)]Cl₃ 128 IV

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-di(2-methoxyethyl)amino-2,2′-bipyridine)]Cl₃  −86 V

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-(3-methoxypropyl)amino-2,2′-bipyridine)]Cl₃  −97 VI

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-diethylamino-2,2′-bipyridine)]Cl₃ −120 VII

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4,4′-dimethyl-2,2′-bipyridine)]Cl₃  32 VIII

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-(6-hydroxyhexyl)amino-2,2′-bipyridine)]Cl₃ −100 IX

  [Os(1,1′-dimethyl-2,2′-biimidazole)₂(4-(6-aminohexyl)amino-2,2′-bipyridine)]Cl₃  −93

Electrochemical Sensors

Generally, embodiments of the invention relate to methods and devicesfor detecting at least one analyte, such as glucose, in body fluid.Embodiments relate to the continuous and/or automatic in vivo monitoringof the level of one or more analytes using a continuous analytemonitoring system that includes an analyte sensor—at least a portion ofwhich is to be positioned beneath a skin surface of a user for a periodof time and/or the discrete monitoring (in vitro monitoring) of one ormore analytes using an in vitro blood glucose (“BG”) meter and ananalyte test strip. Embodiments include combined or combinable devices,systems and methods and/or transferring data between an in vivocontinuous system and a BG meter system.

An electrochemical sensor that includes the cationic polymer basedsensing layer can be formed on a substrate. The sensor may also includeat least one counter electrode (or counter/reference electrode) and/orat least one reference electrode. An “electrochemical sensor” is adevice configured to detect the presence and/or measure the level of ananalyte in a sample, via an electrochemical oxidation or reductionreaction on the sensor, or via a sequence of chemical reactions where atleast one of the chemical reactions is an electrochemical oxidation orreduction reactions on the sensor. These reactions are transduced to anelectrical signal that can be correlated to an amount, concentration, orlevel of an analyte in the sample.

Accordingly, embodiments include analyte monitoring devices and systemsthat include an analyte sensor—at least a portion of which ispositionable beneath the skin of the user—for the in vivo detection, ofan analyte, such as glucose, lactate, and the like, in a body fluid.Embodiments include wholly implantable analyte sensors and analytesensors in which only a portion of the sensor is positioned under theskin and a portion of the sensor resides above the skin, e.g., forcontact to a transmitter, receiver, transceiver, processor, etc. Thesensor may be, for example, subcutaneously positionable in a patient forthe continuous or periodic monitoring of a level of an analyte in apatient's interstitial fluid. For the purposes of this description,continuous monitoring and periodic monitoring will be usedinterchangeably, unless noted otherwise. The sensor response may becorrelated and/or converted to analyte levels in blood or other fluids.In certain embodiments, an analyte sensor may be positioned in contactwith interstitial fluid to detect the level of glucose, which detectedglucose may be used to infer the glucose level in the patient'sbloodstream. Analyte sensors may be insertable into a vein, artery, orother portion of the body containing fluid. Embodiments of the analytesensors of the subject invention having a cationic polymer based sensinglayer may be configured for monitoring the level of the analyte over atime period which may range from minutes, hours, days, weeks, to months,or longer.

Of interest are analyte sensors, such as glucose sensors, having acationic polymer based sensing layer, that are capable of in vivodetection of an analyte for about one hour or more, e.g., about a fewhours or more, e.g., about a few days of more, e.g., about three or moredays, e.g., about five days or more, e.g., about seven days or more,e.g., about several weeks or at least one month or more. Future analytelevels may be predicted based on information obtained, e.g., the currentanalyte level at time t₀, the rate of change of the analyte, etc.Predictive alarms may notify the user of a predicted analyte levels thatmay be of concern in advance of the user's analyte level reaching thefuture level. This provides the user an opportunity to take correctiveaction.

FIG. 1 shows a data monitoring and management system such as, forexample, an analyte (e.g., glucose) monitoring system 100 in accordancewith certain embodiments. Embodiments of the subject invention arefurther described primarily with respect to glucose monitoring devicesand systems, and methods of glucose detection, for convenience only andsuch description is in no way intended to limit the scope of theinvention. It is to be understood that the analyte monitoring system maybe configured to monitor a variety of analytes at the same time or atdifferent times.

Analytes that may be monitored include, but are not limited to, acetylcholine, amylase, bilirubin, cholesterol, chorionic gonadotropin,creatine kinase (e.g., CK-MB), creatine, creatinine, DNA, fructosamine,glucose, glutamine, growth hormones, hormones, ketone bodies, lactate,peroxide, prostate-specific antigen, prothrombin, RNA, thyroidstimulating hormone, and troponin. The concentration of drugs, such as,for example, antibiotics (e.g., gentamicin, vancomycin, and the like),digitoxin, digoxin, drugs of abuse, theophylline, and warfarin, may alsobe monitored. In those embodiments that monitor more than one analyte,the analytes may be monitored at the same or different times.

The analyte monitoring system 100 includes a sensor 101, a dataprocessing unit 102 connectable to the sensor 101, and a primaryreceiver unit 104 which is configured to communicate with the dataprocessing unit 102 via a communication link 103. In certainembodiments, the primary receiver unit 104 may be further configured totransmit data to a data processing terminal 105 to evaluate or otherwiseprocess or format data received by the primary receiver unit 104. Thedata processing terminal 105 may be configured to receive data directlyfrom the data processing unit 102 via a communication link which mayoptionally be configured for bi-directional communication. Further, thedata processing unit 102 may include a transmitter or a transceiver totransmit and/or receive data to and/or from the primary receiver unit104 and/or the data processing terminal 105 and/or optionally thesecondary receiver unit 106.

Also shown in FIG. 1 is an optional secondary receiver unit 106 which isoperatively coupled to the communication link and configured to receivedata transmitted from the data processing unit 102. The secondaryreceiver unit 106 may be configured to communicate with the primaryreceiver unit 104, as well as the data processing terminal 105. Thesecondary receiver unit 106 may be configured for bi-directionalwireless communication with each of the primary receiver unit 104 andthe data processing terminal 105. As discussed in further detail below,in certain embodiments the secondary receiver unit 106 may be ade-featured receiver as compared to the primary receiver, i.e., thesecondary receiver may include a limited or minimal number of functionsand features as compared with the primary receiver unit 104. As such,the secondary receiver unit 106 may include a smaller (in one or more,including all, dimensions), compact housing or embodied in a device suchas a wrist watch, arm band, etc., for example. Alternatively, thesecondary receiver unit 106 may be configured with the same orsubstantially similar functions and features as the primary receiverunit 104. The secondary receiver unit 106 may include a docking portionto be mated with a docking cradle unit for placement by, e.g., thebedside for night time monitoring, and/or a bi-directional communicationdevice. A docking cradle may recharge a powers supply.

Only one sensor 101, data processing unit 102 and data processingterminal 105 are shown in the embodiment of the analyte monitoringsystem 100 illustrated in FIG. 1. However, it will be appreciated by oneof ordinary skill in the art that the analyte monitoring system 100 mayinclude more than one sensor 101 and/or more than one data processingunit 102, and/or more than one data processing terminal 105. Multiplesensors may be positioned in a patient for analyte monitoring at thesame or different times. In certain embodiments, analyte informationobtained by a first positioned sensor may be employed as a comparison toanalyte information obtained by a second sensor. This may be useful toconfirm or validate analyte information obtained from one or both of thesensors. Such redundancy may be useful if analyte information iscontemplated in critical therapy-related decisions. In certainembodiments, a first sensor may be used to calibrate a second sensor.

The analyte monitoring system 100 may be a continuous monitoring system,or semi-continuous, or a discrete monitoring system. In amulti-component environment, each component may be configured to beuniquely identified by one or more of the other components in the systemso that communication conflict may be readily resolved between thevarious components within the analyte monitoring system 100. Forexample, unique IDs, communication channels, and the like, may be used.

In certain embodiments, the sensor 101 is physically positioned in or onthe body of a user whose analyte level is being monitored. The sensor101 may be configured to at least periodically sample the analyte levelof the user and convert the sampled analyte level into a correspondingsignal for transmission by the data processing unit 102. The dataprocessing unit 102 is coupleable to the sensor 101 so that both devicesare positioned in or on the user's body, with at least a portion of theanalyte sensor 101 positioned transcutaneously. The data processing unitmay include a fixation element such as adhesive or the like to secure itto the user's body. A mount (not shown) attachable to the user andmateable with the unit 102 may be used. For example, a mount may includean adhesive surface. The data processing unit 102 performs dataprocessing functions, where such functions may include but are notlimited to, filtering and encoding of data signals, each of whichcorresponds to a sampled analyte level of the user, for transmission tothe primary receiver unit 104 via the communication link 103. In oneembodiment, the sensor 101 or the data processing unit 102 or a combinedsensor/data processing unit may be wholly implantable under the skinlayer of the user.

In certain embodiments, the primary receiver unit 104 may include ananalog interface section including and RF receiver and an antenna thatis configured to communicate with the data processing unit 102 via thecommunication link 103, and a data processing section for processing thereceived data from the data processing unit 102 such as data decoding,error detection and correction, data clock generation, data bitrecovery, etc., or any combination thereof

In operation, the primary receiver unit 104 in certain embodiments isconfigured to synchronize with the data processing unit 102 to uniquelyidentify the data processing unit 102, based on, for example, anidentification information of the data processing unit 102, andthereafter, to periodically receive signals transmitted from the dataprocessing unit 102 associated with the monitored analyte levelsdetected by the sensor 101.

Referring again to FIG. 1, the data processing terminal 105 may includea personal computer, a portable computer such as a laptop or a handhelddevice (e.g., personal digital assistants (PDAs), telephone such as acellular phone (e.g., a multimedia and Internet-enabled mobile phonesuch as an iPhone™ or similar phone), mp3 player, pager, and the like),drug delivery device, each of which may be configured for datacommunication with the receiver via a wired or a wireless connection.Additionally, the data processing terminal 105 may further be connectedto a data network (not shown) for storing, retrieving, updating, and/oranalyzing data corresponding to the detected analyte level of the user.

The data processing terminal 105 may include an infusion device such asan insulin infusion pump or the like, which may be configured toadminister insulin to patients, and which may be configured tocommunicate with the primary receiver unit 104 for receiving, amongothers, the measured analyte level. Alternatively, the primary receiverunit 104 may be configured to integrate an infusion device therein sothat the primary receiver unit 104 is configured to administer insulin(or other appropriate drug) therapy to patients, for example, foradministering and modifying basal profiles, as well as for determiningappropriate boluses for administration based on, among others, thedetected analyte levels received from the data processing unit 102. Aninfusion device may be an external device or an internal device (whollyimplantable in a user).

In certain embodiments, the data processing terminal 105, which mayinclude an insulin pump, may be configured to receive the analytesignals from the data processing unit 102, and thus, incorporate thefunctions of the primary receiver unit 104 including data processing formanaging the patient's insulin therapy and analyte monitoring. Incertain embodiments, the communication link 103 as well as one or moreof the other communication interfaces shown in FIG. 1, may use one ormore of: an RF communication protocol, an infrared communicationprotocol, a Bluetooth enabled communication protocol, an 802.11xwireless communication protocol, or an equivalent wireless communicationprotocol which would allow secure, wireless communication of severalunits (for example, per HIPPA requirements), while avoiding potentialdata collision and interference.

FIG. 2 shows a block diagram of an embodiment of a data processing unitof the data monitoring and detection system shown in FIG. 1. User inputand/or interface components may be included or a data processing unitmay be free of user input and/or interface components. In certainembodiments, one or more application-specific integrated circuits (ASIC)may be used to implement one or more functions or routins associatedwith the operations of the data processing unit (and/or receiver unit)using for example one or more state machines and buffers.

As can be seen in the embodiment of FIG. 2, the sensor unit 101 (FIG. 1)includes four contacts, three of which are electrodes—work electrode (W)210, reference electrode (R) 212, and counter electrode (C) 213, eachoperatively coupled to the analog interface 201 of the data processingunit 102. This embodiment also shows optional guard contact (G) 211.Fewer or greater electrodes may be employed. For example, the counterand reference electrode functions may be served by a singlecounter/reference electrode, there may be more than one workingelectrode and/or reference electrode and/or counter electrode, etc.

FIG. 3 is a block diagram of an embodiment of a receiver/monitor unitsuch as the primary receiver unit 104 of the data monitoring andmanagement system shown in FIG. 1. The primary receiver unit 104includes one or more of: a blood glucose test strip interface 301, an RFreceiver 302, an input 303, a temperature detection section 304, and aclock 305, each of which is operatively coupled to a processing andstorage section 307. The primary receiver unit 104 also includes a powersupply 306 operatively coupled to a power conversion and monitoringsection 308. Further, the power conversion and monitoring section 308 isalso coupled to the receiver processor 307. Moreover, also shown are areceiver serial communication section 309, and an output 310, eachoperatively coupled to the processing and storage unit 307. The receivermay include user input and/or interface components or may be free ofuser input and/or interface components.

In certain embodiments, the test strip interface 301 includes a glucoselevel testing portion to receive a blood (or other body fluid sample)glucose test or information related thereto. For example, the interfacemay include a test strip port to receive a glucose test strip. Thedevice may determine the glucose level of the test strip, and optionallydisplay (or otherwise notice) the glucose level on the output 310 of theprimary receiver unit 104. Any suitable test strip may be employed,e.g., test strips that only require a very small amount (e.g., onemicroliter or less, e.g., 0.5 microliter or less, e.g., 0.1 microliteror less), of applied sample to the strip in order to obtain accurateglucose information, e.g. FreeStyle® blood glucose test strips fromAbbott Diabetes Care, Inc. Glucose information obtained by the in vitroglucose testing device may be used for a variety of purposes,computations, etc. For example, the information may be used to calibratesensor 101, confirm results of the sensor 101 to increase the confidencethereof (e.g., in instances in which information obtained by sensor 101is employed in therapy related decisions), etc.

In further embodiments, the data processing unit 102 and/or the primaryreceiver unit 104 and/or the secondary receiver unit 105, and/or thedata processing terminal/infusion section 105 may be configured toreceive the blood glucose value wirelessly over a communication linkfrom, for example, a blood glucose meter. In further embodiments, a usermanipulating or using the analyte monitoring system 100 (FIG. 1) maymanually input the blood glucose value using, for example, a userinterface (for example, a keyboard, keypad, voice commands, and thelike) incorporated in the one or more of the data processing unit 102,the primary receiver unit 104, secondary receiver unit 105, or the dataprocessing terminal/infusion section 105.

Additional detailed descriptions are provided in U.S. Pat. Nos.5,262,035; 5,264,104; 5,262,305; 5,320,715; 5,593,852; 6,175,752;6,650,471; 6,746, 582, and in application Ser. No. 10/745,878 filed Dec.26, 2003 entitled “Continuous Glucose Monitoring System and Methods ofUse”, each of which is incorporated herein by reference.

FIG. 4 schematically shows an embodiment of an analyte sensor inaccordance with the embodiments of the invention. This sensor embodimentincludes electrodes 401, 402 and 403 on a base 404. Electrodes (and/orother features) may be applied or otherwise processed using any suitabletechnology, e.g., chemical vapor deposition (CVD), physical vapordeposition, sputtering, reactive sputtering, printing, coating, ablating(e.g., laser ablation), painting, dip coating, etching, and the like.Materials include but are not limited to aluminum, carbon (such asgraphite), cobalt, copper, gallium, gold, indium, iridium, iron, lead,magnesium, mercury (as an amalgam), nickel, niobium, osmium, palladium,platinum, rhenium, rhodium, selenium, silicon (e.g., dopedpolycrystalline silicon), silver, tantalum, tin, titanium, tungsten,uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys,oxides, or metallic compounds of these elements.

The sensor may be wholly implantable in a user or may be configured sothat only a portion is positioned within (internal) a user and anotherportion outside (external) a user. For example, the sensor 400 mayinclude a portion positionable above a surface of the skin 410, and aportion positioned below the skin. In such embodiments, the externalportion may include contacts (connected to respective electrodes of thesecond portion by traces) to connect to another device also external tothe user such as a transmitter unit. While the embodiment of FIG. 4shows three electrodes side-by-side on the same surface of base 404,other configurations are contemplated, e.g., fewer or greaterelectrodes, some or all electrodes on different surfaces of the base orpresent on another base, some or all electrodes stacked together,electrodes of differing materials and dimensions, etc.

FIG. 5A shows a perspective view of an embodiment of an electrochemicalanalyte sensor 500 having a first portion (which in this embodiment maybe characterized as a major portion) positionable above a surface of theskin 510, and a second portion (which in this embodiment may becharacterized as a minor portion) that includes an insertion tip 530positionable below the skin, e.g., penetrating through the skin andinto, e.g., the subcutaneous space 520, in contact with the user'sbiofluid such as interstitial fluid. Contact portions of a workingelectrode 501, a reference electrode 502, and a counter electrode 503are positioned on the portion of the sensor 500 situated above the skinsurface 510. Working electrode 501, a reference electrode 502, and acounter electrode 503 are shown at the second section and particularlyat the insertion tip 530. Traces may be provided from the electrode atthe tip to the contact, as shown in FIG. 5A. It is to be understood thatgreater or fewer electrodes may be provided on a sensor. For example, asensor may include more than one working electrode and/or the counterand reference electrodes may be a single counter/reference electrode,etc.

FIG. 5B shows a cross sectional view of a portion of the sensor 500 ofFIG. 5A. The electrodes 501, 502 and 503, of the sensor 500 as well asthe substrate and the dielectric layers are provided in a layeredconfiguration or construction. For example, as shown in FIG. 5B, in oneaspect, the sensor 500 (such as the sensor unit 101 FIG. 1), includes asubstrate layer 504, and a first conducting layer 501 such as carbon,gold, etc., disposed on at least a portion of the substrate layer 504,and which may provide the working electrode. Also shown disposed on atleast a portion of the first conducting layer 501 is a sensing layer508.

A first insulation layer such as a first dielectric layer 505 isdisposed or layered on at least a portion of the first conducting layer501, and further, a second conducting layer 509 may be disposed orstacked on top of at least a portion of the first insulation layer (ordielectric layer) 505. As shown in FIG. 5B, the second conducting layer509 may provide the reference electrode 502, as described herein havingan extended lifetime, which includes a layer of redox polymer asdescribed herein.

A second insulation layer 506 such as a dielectric layer in oneembodiment may be disposed or layered on at least a portion of thesecond conducting layer 509. Further, a third conducting layer 503 mayprovide the counter electrode 503. It may be disposed on at least aportion of the second insulation layer 506. Finally, a third insulationlayer may be disposed or layered on at least a portion of the thirdconducting layer 503. In this manner, the sensor 500 may be layered suchthat at least a portion of each of the conducting layers is separated bya respective insulation layer (for example, a dielectric layer). Theembodiment of FIGS. 5A and 5B show the layers having different lengths.Some or all of the layers may have the same or different lengths and/orwidths.

In certain embodiments, some or all of the electrodes 501, 502, 503 maybe provided on the same side of the substrate 504 in the layeredconstruction as described above, or alternatively, may be provided in aco-planar manner such that two or more electrodes may be positioned onthe same plane (e.g., side-by side (e.g., parallel) or angled relativeto each other) on the substrate 504. For example, co-planar electrodesmay include a suitable spacing there between and/or include dielectricmaterial or insulation material disposed between the conductinglayers/electrodes. Furthermore, in certain embodiments one or more ofthe electrodes 501, 502, 503 may be disposed on opposing sides of thesubstrate 504. In such embodiments, contact pads may be one the same ordifferent sides of the substrate. For example, an electrode may be on afirst side and its respective contact may be on a second side, e.g., atrace connecting the electrode and the contact may traverse through thesubstrate.

As noted above, analyte sensors may include an analyte-responsive enzymeto provide a sensing component or sensing layer. Some analytes, such asoxygen, can be directly electrooxidized or electroreduced on a sensor,and more specifically at least on a working electrode of a sensor. Otheranalytes, such as glucose and lactate, require the presence of at leastone electron transfer agent and/or at least one catalyst to facilitatethe electrooxidation or electroreduction of the analyte. Catalysts mayalso be used for those analyte, such as oxygen, that can be directlyelectrooxidized or electroreduced on the working electrode. For theseanalytes, each working electrode includes a sensing layer (see forexample sensing layer 408 of FIG. 5B) proximate to or on a surface of aworking electrode. In many embodiments, a sensing layer is formed nearor on only a small portion of at least a working electrode.

The sensing layer includes one or more components designed to facilitatethe electrochemical oxidation or reduction of the analyte. The sensinglayer may include, for example, a catalyst to catalyze a reaction of theanalyte and produce a response at the working electrode, an electrontransfer agent to transfer electrons between the analyte and the workingelectrode (or other component), or both.

A variety of different sensing layer configurations may be used. Incertain embodiments, the sensing layer is deposited on the conductivematerial of a working electrode. The sensing layer may extend beyond theconductive material of the working electrode. In some cases, the sensinglayer may also extend over other electrodes, e.g., over the counterelectrode and/or reference electrode (or counter/reference is provided).

A sensing layer that is in direct contact with the working electrode maycontain an electron transfer agent to transfer electrons directly orindirectly between the analyte and the working electrode, and/or acatalyst to facilitate a reaction of the analyte. For example, aglucose, lactate, or oxygen electrode may be formed having a sensinglayer which contains a catalyst, such as glucose oxidase, lactateoxidase, or laccase, respectively, and an electron transfer agent thatfacilitates the electrooxidation of the glucose, lactate, or oxygen,respectively.

In other embodiments the sensing layer is not deposited directly on theworking electrode. Instead, the sensing layer 64 may be spaced apartfrom the working electrode, and separated from the working electrode,e.g., by a separation layer. A separation layer may include one or moremembranes or films or a physical distance. In addition to separating theworking electrode from the sensing layer the separation layer may alsoact as a mass transport limiting layer and/or an interferent eliminatinglayer and/or a biocompatible layer.

In certain embodiments which include more than one working electrode,one or more of the working electrodes may not have a correspondingsensing layer, or may have a sensing layer which does not contain one ormore components (e.g., an electron transfer agent and/or catalyst)needed to electrolyze the analyte. Thus, the signal at this workingelectrode may correspond to background signal which may be removed fromthe analyte signal obtained from one or more other working electrodesthat are associated with fully-functional sensing layers by, forexample, subtracting the signal.

In certain embodiments, the sensing layer includes one or more electrontransfer agents. Electron transfer agents that may be employed areelectroreducible and electrooxidizable ions or molecules having redoxpotentials that are a few hundred millivolts above or below the redoxpotential of the standard calomel electrode (SCE). The electron transferagent may be organic, organometallic, or inorganic. Examples of organicredox species are quinones and species that in their oxidized state havequinoid structures, such as Nile blue and indophenol. Examples oforganometallic redox species are metallocenes such as ferrocene.Examples of inorganic redox species are hexacyanoferrate (III),ruthenium hexamine etc.

In certain embodiments, electron transfer agents have structures orcharges which prevent or substantially reduce the diffusional loss ofthe electron transfer agent during the period of time that the sample isbeing analyzed. For example, electron transfer agents include but arenot limited to a redox species, e.g., bound to a polymer which can inturn be disposed on or near the working electrode. The bond between theredox species and the polymer may be covalent, coordinative, or ionic.Although any organic, organometallic or inorganic redox species may bebound to a polymer and used as an electron transfer agent, in certainembodiments the redox species is a transition metal compound or complex,e.g., osmium, ruthenium, iron, and cobalt compounds or complexes. Itwill be recognized that many redox species described for use with apolymeric component may also be used, without a polymeric component.

One type of polymeric electron transfer agent contains a redox speciescovalently bound in a polymeric composition. An example of this type ofmediator is poly(vinylferrocene). Another type of electron transferagent contains an ionically-bound redox species. This type of mediatormay include a charged polymer coupled to an oppositely charged redoxspecies. Examples of this type of mediator include a negatively chargedpolymer coupled to a positively charged redox species such as an osmiumor ruthenium polypyridyl cation. Another example of an ionically-boundmediator is a positively charged polymer such as quaternizedpoly(4-vinyl pyridine) or poly(l-vinyl imidazole) coupled to anegatively charged redox species such as ferricyanide or ferrocyanide.In other embodiments, electron transfer agents include a redox speciescoordinatively bound to a polymer. For example, the mediator may beformed by coordination of an osmium or cobalt 2,2′-bipyridyl complex topoly(1-vinyl imidazole) or poly(4-vinyl pyridine).

Suitable electron transfer agents are osmium transition metal complexeswith one or more ligands, each ligand having a nitrogen-containingheterocycle such as 2,2′-bipyridine, 1,10-phenanthroline, 1-methyl,2-pyridyl biimidazole, or derivatives thereof. The electron transferagents may also have one or more ligands covalently bound in a polymer,each ligand having at least one nitrogen-containing heterocycle, such aspyridine, imidazole, or derivatives thereof. One example of an electrontransfer agent includes (a) a polymer or copolymer having pyridine orimidazole functional groups and (b) osmium cations complexed with twoligands, each ligand containing 2,2′-bipyridine, 1,10-phenanthroline, orderivatives thereof, the two ligands not necessarily being the same.Some derivatives of 2,2′-bipyridine for complexation with the osmiumcation include but are not limited to 4,4′-dimethyl-2,2′-bipyridine andmono-, di-, and polyalkoxy-2,2′-bipyridines, such as4,4′-dimethoxy-2,2′-bipyridine. Derivatives of 1,10-phenanthroline forcomplexation with the osmium cation include but are not limited to4,7-dimethyl-1,10-phenanthroline and mono, di-, andpolyalkoxy-1,10-phenanthrolines, such as4,7-dimethoxy-1,10-phenanthroline. Polymers for complexation with theosmium cation include but are not limited to polymers and copolymers ofpoly(l-vinyl imidazole) (referred to as “PVI”) and poly(4-vinylpyridine) (referred to as “PVP”). Suitable copolymer substituents ofpoly(l-vinyl imidazole) include acrylonitrile, acrylamide, andsubstituted or quaternized N-vinyl imidazole, e.g., electron transferagents with osmium complexed to a polymer or copolymer of poly(l-vinylimidazole).

Embodiments may employ electron transfer agents having a redox potentialranging from about −200 mV to about +200 mV versus the standard calomelelectrode (SCE). The sensing layer may also include a catalyst which iscapable of catalyzing a reaction of the analyte. The catalyst may also,in some embodiments, act as an electron transfer agent. One example of asuitable catalyst is an enzyme which catalyzes a reaction of theanalyte. For example, a catalyst, such as a glucose oxidase, glucosedehydrogenase (e.g., pyrroloquinoline quinone (PQQ), dependent glucosedehydrogenase, flavine adenine dinucleotide (FAD) dependent glucosedehydrogenase, or nicotinamide adenine dinucleotide (NAD) dependentglucose dehydrogenase), may be used when the analyte of interest isglucose. A lactate oxidase or lactate dehydrogenase may be used when theanalyte of interest is lactate. Laccase may be used when the analyte ofinterest is oxygen or when oxygen is generated or consumed in responseto a reaction of the analyte.

The sensing layer may also include a catalyst which is capable ofcatalyzing a reaction of the analyte. The catalyst may also, in someembodiments, act as an electron transfer agent. One example of asuitable catalyst is an enzyme which catalyzes a reaction of theanalyte. For example, a catalyst, such as a glucose oxidase, glucosedehydrogenase (e.g., pyrroloquinoline quinone (PQQ), dependent glucosedehydrogenase or oligosaccharide dehydrogenase, flavine adeninedinucleotide (FAD) dependent glucose dehydrogenase, nicotinamide adeninedinucleotide (NAD) dependent glucose dehydrogenase), may be used whenthe analyte of interest is glucose. A lactate oxidase or lactatedehydrogenase may be used when the analyte of interest is lactate.Laccase may be used when the analyte of interest is oxygen or whenoxygen is generated or consumed in response to a reaction of theanalyte.

In certain embodiments, a catalyst may be attached to a polymer, crosslinking the catalyst with another electron transfer agent (which, asdescribed above, may be polymeric. A second catalyst may also be used incertain embodiments. This second catalyst may be used to catalyze areaction of a product compound resulting from the catalyzed reaction ofthe analyte. The second catalyst may operate with an electron transferagent to electrolyze the product compound to generate a signal at theworking electrode. Alternatively, a second catalyst may be provided inan interferent-eliminating layer to catalyze reactions that removeinterferents.

In certain embodiments, the sensor includes the cationic polymer basedsensing layer sensing layer and works at a gentle oxidizing potential,e.g., a potential of about +40 mV vs. Ag/AgCl. This sensing layer uses,for example, an osmium (Os)-based mediator designed for low potentialoperation and is stabilized by the cationic polymer. Accordingly, incertain embodiments the sensing element is a redox active component thatincludes (1) Osmium-based mediator molecules that include (bidente)ligands, and (2) glucose oxidase enzyme molecules. These twoconstituents are combined together with a cationic polymer.

A mass transport limiting layer (not shown), e.g., an analyte fluxmodulating layer, may be included with the sensor to act as adiffusion-limiting barrier to reduce the rate of mass transport of theanalyte, for example, glucose or lactate, into the region around theworking electrodes. The mass transport limiting layers are useful inlimiting the flux of an analyte to a working electrode in anelectrochemical sensor so that the sensor is linearly responsive over alarge range of analyte concentrations and is easily calibrated. Masstransport limiting layers may include polymers and may be biocompatible.A mass transport limiting layer may provide many functions, e.g.,biocompatibility and/or interferent-eliminating, etc.

In certain embodiments, a mass transport limiting layer is a membranecomposed of crosslinked polymers containing heterocyclic nitrogengroups, such as polymers of polyvinylpyridine and polyvinylimidazole.Embodiments also include membranes that are made of a polyurethane, orpolyether urethane, or chemically related material, or membranes thatare made of silicone, and the like.

A membrane may be formed by crosslinking in situ a polymer, modifiedwith a zwitterionic moiety, a non-pyridine copolymer component, andoptionally another moiety that is either hydrophilic or hydrophobic,and/or has other desirable properties, in an alcohol-buffer solution.The modified polymer may be made from a precursor polymer containingheterocyclic nitrogen groups. For example, a precursor polymer may bepolyvinylpyridine or polyvinylimidazole. Optionally, hydrophilic orhydrophobic modifiers may be used to “fine-tune” the permeability of theresulting membrane to an analyte of interest. Optional hydrophilicmodifiers, such as poly(ethylene glycol), hydroxyl or polyhydroxylmodifiers, may be used to enhance the biocompatibility of the polymer orthe resulting membrane.

A membrane may be formed in situ by applying an alcohol-buffer solutionof a crosslinker and a modified polymer over an enzyme-containingsensing layer and allowing the solution to cure for about one to twodays or other appropriate time period. The crosslinker-polymer solutionmay be applied to the sensing layer by placing a droplet or droplets ofthe solution on the sensor, by dipping the sensor into the solution, orthe like. Generally, the thickness of the membrane is controlled by theconcentration of the solution, by the number of droplets of the solutionapplied, by the number of times the sensor is dipped in the solution, orby any combination of these factors. A membrane applied in this mannermay have any combination of the following functions: (1) mass transportlimitation, i.e., reduction of the flux of analyte that can reach thesensing layer, (2) biocompatibility enhancement, or (3) interferentreduction.

In certain embodiments, the sensing system detects hydrogen peroxide toinfer glucose levels. For example, a hydrogen peroxide-detecting sensormay be constructed in which a sensing layer includes enzyme such asglucose oxides, glucose dehydrogensae, or the like, and is positionedproximate to the working electrode. The sensing layer may be covered byone or more layers, e.g., a membrane that is selectively permeable toglucose. Once the glucose passes through the membrane, it is oxidized bythe enzyme and reduced glucose oxidase can then be oxidized by reactingwith molecular oxygen to produce hydrogen peroxide.

Certain embodiments include a hydrogen peroxide-detecting sensorconstructed from a sensing layer prepared by combining together, forexample: (1) a redox mediator having a transition metal complex such asa Os polypyridyl complexes with oxidation potentials of about +200 mVvs. SCE, (2) cationic polymer, and (3) periodate oxidized horseradishperoxidase (HRP). Such a sensor functions in a reductive mode; theworking electrode is controlled at a potential negative to that of theOs complex, resulting in mediated reduction of hydrogen peroxide throughthe HRP catalyst.

In another example, a potentiometric sensor can be constructed asfollows. A glucose-sensing layer is constructed by combining together(1) a redox mediator having a transition metal complex such as a Ospolypyridyl complexes with oxidation potentials from about −200 mV to+200 mV vs. SCE, and (2) cationic polymer, and (3) glucose oxidase. Thissensor can then be used in a potentiometric mode, by exposing the sensorto a glucose containing solution, under conditions of zero current flow,and allowing the ratio of reduced/oxidized Os to reach an equilibriumvalue. The reduced/oxidized Os ratio varies in a reproducible way withthe glucose concentration, and will cause the electrode's potential tovary in a similar way.

The substrate may be formed using a variety of non-conducting materials,including, for example, polymeric or plastic materials and ceramicmaterials. Suitable materials for a particular sensor may be determined,at least in part, based on the desired use of the sensor and propertiesof the materials.

In some embodiments, the substrate is flexible. For example, if thesensor is configured for implantation into a patient, then the sensormay be made flexible (although rigid sensors may also be used forimplantable sensors) to reduce pain to the patient and damage to thetissue caused by the implantation of and/or the wearing of the sensor. Aflexible substrate often increases the patient's comfort and allows awider range of activities. Suitable materials for a flexible substrateinclude, for example, non-conducting plastic or polymeric materials andother non-conducting, flexible, deformable materials. Examples of usefulplastic or polymeric materials include thermoplastics such aspolycarbonates, polyesters (e.g., Mylar™ and polyethylene terephthalate(PET)), polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides,polyimides, or copolymers of these thermoplastics, such as PETG(glycol-modified polyethylene terephthalate).

In other embodiments, the sensors are made using a relatively rigidsubstrate to, for example, provide structural support against bending orbreaking Examples of rigid materials that may be used as the substrateinclude poorly conducting ceramics, such as aluminum oxide and silicondioxide. One advantage of an implantable sensor having a rigid substrateis that the sensor may have a sharp point and/or a sharp edge to aid inimplantation of a sensor without an additional insertion device.

It will be appreciated that for many sensors and sensor applications,both rigid and flexible sensors will operate adequately. The flexibilityof the sensor may also be controlled and varied along a continuum bychanging, for example, the composition and/or thickness of thesubstrate.

In addition to considerations regarding flexibility, it is oftendesirable that implantable sensors should have a substrate which isphysiologically harmless, for example, a substrate approved by aregulatory agency or private institution for in vivo use.

The sensor may include optional features to facilitate insertion of animplantable sensor. For example, the sensor may be pointed at the tip toease insertion. In addition, the sensor may include a barb which assistsin anchoring the sensor within the tissue of the patient duringoperation of the sensor. However, the barb is typically small enough sothat little damage is caused to the subcutaneous tissue when the sensoris removed for replacement.

An implantable sensor may also, optionally, have an anticlotting agentdisposed on a portion the substrate which is implanted into a patient.This anticlotting agent may reduce or eliminate the clotting of blood orother body fluid around the sensor, particularly after insertion of thesensor. Blood clots may foul the sensor or irreproducibly reduce theamount of analyte which diffuses into the sensor. Examples of usefulanticlotting agents include heparin and tissue plasminogen activator(TPA), as well as other known anticlotting agents.

The anticlotting agent may be applied to at least a portion of that partof the sensor that is to be implanted. The anticlotting agent may beapplied, for example, by bath, spraying, brushing, or dipping. Theanticlotting agent is allowed to dry on the sensor. The anticlottingagent may be immobilized on the surface of the sensor or it may beallowed to diffuse away from the sensor surface. Typically, thequantities of anticlotting agent disposed on the sensor are far belowthe amounts typically used for treatment of medical conditions involvingblood clots and, therefore, have only a limited, localized effect.

Insertion Device

An insertion device can be used to subcutaneously insert the sensor intothe patient. The insertion device is typically formed using structurallyrigid materials, such as metal or rigid plastic. Exemplary materialsinclude stainless steel and ABS (acrylonitrile-butadiene-styrene)plastic. In some embodiments, the insertion device is pointed and/orsharp at the tip to facilitate penetration of the skin of the patient. Asharp, thin insertion device may reduce pain felt by the patient uponinsertion of the sensor. In other embodiments, the tip of the insertiondevice has other shapes, including a blunt or flat shape. Theseembodiments may be particularly useful when the insertion device doesnot penetrate the skin but rather serves as a structural support for thesensor as the sensor is pushed into the skin.

Sensor Control Unit

The sensor control unit can be integrated in the sensor, part or all ofwhich is subcutaneously implanted or it can be configured to be placedon the skin of a patient. The sensor control unit is optionally formedin a shape that is comfortable to the patient and which may permitconcealment, for example, under a patient's clothing. The thigh, leg,upper arm, shoulder, or abdomen are convenient parts of the patient'sbody for placement of the sensor control unit to maintain concealment.However, the sensor control unit may be positioned on other portions ofthe patient's body. One embodiment of the sensor control unit has athin, oval shape to enhance concealment. However, other shapes and sizesmay be used.

The particular profile, as well as the height, width, length, weight,and volume of the sensor control unit may vary and depends, at least inpart, on the components and associated functions included in the sensorcontrol unit. In general, the sensor control unit includes a housingtypically formed as a single integral unit that rests on the skin of thepatient. The housing typically contains most or all of the electroniccomponents of the sensor control unit.

The housing of the sensor control unit may be formed using a variety ofmaterials, including, for example, plastic and polymeric materials,particularly rigid thermoplastics and engineering thermoplastics.Suitable materials include, for example, polyvinyl chloride,polyethylene, polypropylene, polystyrene, ABS polymers, and copolymersthereof. The housing of the sensor control unit may be formed using avariety of techniques including, for example, injection molding,compression molding, casting, and other molding methods. Hollow orrecessed regions may be formed in the housing of the sensor controlunit. The electronic components of the sensor control unit and/or otheritems, such as a battery or a speaker for an audible alarm, may beplaced in the hollow or recessed areas.

The sensor control unit is typically attached to the skin of thepatient, for example, by adhering the sensor control unit directly tothe skin of the patient with an adhesive provided on at least a portionof the housing of the sensor control unit which contacts the skin or bysuturing the sensor control unit to the skin through suture openings inthe sensor control unit.

When positioned on the skin of a patient, the sensor and the electroniccomponents within the sensor control unit are coupled via conductivecontacts. The one or more working electrodes, counter electrode (orcounter/reference electrode), optional reference electrode, and optionaltemperature probe are attached to individual conductive contacts. Forexample, the conductive contacts are provided on the interior of thesensor control unit. Other embodiments of the sensor control unit havethe conductive contacts disposed on the exterior of the housing. Theplacement of the conductive contacts is such that they are in contactwith the contact pads on the sensor when the sensor is properlypositioned within the sensor control unit.

Sensor Control Unit Electronics

The sensor control unit also typically includes at least a portion ofthe electronic components that operate the sensor and the analytemonitoring device system. The electronic components of the sensorcontrol unit typically include a power supply for operating the sensorcontrol unit and the sensor, a sensor circuit for obtaining signals fromand operating the sensor, a measurement circuit that converts sensorsignals to a desired format, and a processing circuit that, at minimum,obtains signals from the sensor circuit and/or measurement circuit andprovides the signals to an optional transmitter. In some embodiments,the processing circuit may also partially or completely evaluate thesignals from the sensor and convey the resulting data to the optionaltransmitter and/or activate an optional alarm system if the analytelevel exceeds a threshold. The processing circuit often includes digitallogic circuitry.

The sensor control unit may optionally contain a transmitter fortransmitting the sensor signals or processed data from the processingcircuit to a receiver/display unit; a data storage unit for temporarilyor permanently storing data from the processing circuit; a temperatureprobe circuit for receiving signals from and operating a temperatureprobe; a reference voltage generator for providing a reference voltagefor comparison with sensor-generated signals; and/or a watchdog circuitthat monitors the operation of the electronic components in the sensorcontrol unit.

Moreover, the sensor control unit may also include digital and/or analogcomponents utilizing semiconductor devices, such as transistors. Tooperate these semiconductor devices, the sensor control unit may includeother components including, for example, a bias control generator tocorrectly bias analog and digital semiconductor devices, an oscillatorto provide a clock signal, and a digital logic and timing component toprovide timing signals and logic operations for the digital componentsof the circuit.

As an example of the operation of these components, the sensor circuitand the optional temperature probe circuit provide raw signals from thesensor to the measurement circuit. The measurement circuit converts theraw signals to a desired format, using for example, a current-to-voltageconverter, current-to-frequency converter, and/or a binary counter orother indicator that produces a signal proportional to the absolutevalue of the raw signal. This may be used, for example, to convert theraw signal to a format that can be used by digital logic circuits. Theprocessing circuit may then, optionally, evaluate the data and providecommands to operate the electronics.

Calibration

Sensors may be configured to require no system calibration or no usercalibration.

For example, a sensor may be factory calibrated and need not requirefurther calibrating. In certain embodiments, calibration may berequired, but may be done without user intervention, i.e., may beautomatic. In those embodiments in which calibration by the user isrequired, the calibration may be according to a predetermined scheduleor may be dynamic, i.e., the time for which may be determined by thesystem on a real-time basis according to various factors, such as butnot limited to glucose concentration and/or temperature and/or rate ofchange of glucose, etc.

In addition to a transmitter, an optional receiver may be included inthe sensor control unit. In some cases, the transmitter is atransceiver, operating as both a transmitter and a receiver. Thereceiver may be used to receive calibration data for the sensor. Thecalibration data may be used by the processing circuit to correctsignals from the sensor. This calibration data may be transmitted by thereceiver/display unit or from some other source such as a control unitin a doctor's office. In addition, the optional receiver may be used toreceive a signal from the receiver/display units to direct thetransmitter, for example, to change frequencies or frequency bands, toactivate or deactivate the optional alarm system and/or to direct thetransmitter to transmit at a higher rate.

Calibration data may be obtained in a variety of ways. For instance, thecalibration data may simply be factory-determined calibrationmeasurements which can be input into the sensor control unit using thereceiver or may alternatively be stored in a calibration data storageunit within the sensor control unit itself (in which case a receiver maynot be needed). The calibration data storage unit may be, for example, areadable or readable/writeable memory circuit.

Calibration may be accomplished using an in vitro test strip (or otherreference), e.g., a small sample test strip such as a test strip thatrequires less than about 1 microliter of sample (for example FreeStyle®blood glucose monitoring test strips from Abbott Diabetes Care). Forexample, test strips that require less than about 1 nanoliter of samplemay be used. In certain embodiments, a sensor may be calibrated usingonly one sample of body fluid per calibration event. For example, a userneed only lance a body part one time to obtain sample for a calibrationevent (e.g., for a test strip), or may lance more than one time within ashort period of time if an insufficient volume of sample is firstlyobtained. Embodiments include obtaining and using multiple samples ofbody fluid for a given calibration event, where glucose values of eachsample are substantially similar. Data obtained from a given calibrationevent may be used independently to calibrate or combined with dataobtained from previous calibration events, e.g., averaged includingweighted averaged, etc., to calibrate. In certain embodiments, a systemneed only be calibrated once by a user, where recalibration of thesystem is not required.

Alternative or additional calibration data may be provided based ontests performed by a doctor or some other professional or by thepatient. For example, it is common for diabetic individuals to determinetheir own blood glucose concentration using commercially availabletesting kits. The results of this test is input into the sensor controlunit either directly, if an appropriate input device (e.g., a keypad, anoptical signal receiver, or a port for connection to a keypad orcomputer) is incorporated in the sensor control unit, or indirectly byinputting the calibration data into the receiver/display unit andtransmitting the calibration data to the sensor control unit.

Other methods of independently determining analyte levels may also beused to obtain calibration data. This type of calibration data maysupplant or supplement factory-determined calibration values.

In some embodiments of the invention, calibration data may be requiredat periodic intervals, for example, every eight hours, once a day, oronce a week, to confirm that accurate analyte levels are being reported.Calibration may also be required each time a new sensor is implanted orif the sensor exceeds a threshold minimum or maximum value or if therate of change in the sensor signal exceeds a threshold value. In somecases, it may be necessary to wait a period of time after theimplantation of the sensor before calibrating to allow the sensor toachieve equilibrium. In some embodiments, the sensor is calibrated onlyafter it has been inserted. In other embodiments, no calibration of thesensor is needed.

Analyte Monitoring Device

In some embodiments of the invention, the analyte monitoring deviceincludes a sensor control unit and a sensor. In these embodiments, theprocessing circuit of the sensor control unit is able to determine alevel of the analyte and activate an alarm system if the analyte levelexceeds a threshold. The sensor control unit, in these embodiments, hasan alarm system and may also include a display, such as an LCD or LEDdisplay.

A threshold value is exceeded if the datapoint has a value that isbeyond the threshold value in a direction indicating a particularcondition. For example, a datapoint which correlates to a glucose levelof 200 mg/dL exceeds a threshold value for hyperglycemia of 180 mg/dL,because the datapoint indicates that the patient has entered ahyperglycemic state. As another example, a datapoint which correlates toa glucose level of 65 mg/dL exceeds a threshold value for hypoglycemiaof 70 mg/dL because the datapoint indicates that the patient ishypoglycemic as defined by the threshold value. However, a datapointwhich correlates to a glucose level of 75 mg/dL would not exceed thesame threshold value for hypoglycemia because the datapoint does notindicate that particular condition as defined by the chosen thresholdvalue.

An alarm may also be activated if the sensor readings indicate a valuethat is beyond a measurement range of the sensor. For glucose, thephysiologically relevant measurement range is typically about 50 to 250mg/dL, preferably about 40-300 mg/dL and ideally 30-400 mg/dL, ofglucose in the interstitial fluid.

The alarm system may also, or alternatively, be activated when the rateof change or acceleration of the rate of change in analyte levelincrease or decrease reaches or exceeds a threshold rate oracceleration. For example, in the case of a subcutaneous glucosemonitor, the alarm system might be activated if the rate of change inglucose concentration exceeds a threshold value which might indicatethat a hyperglycemic or hypoglycemic condition is likely to occur.

A system may also include system alarms that notify a user of systeminformation such as battery condition, calibration, sensor dislodgment,sensor malfunction, etc. Alarms may be, for example, auditory and/orvisual. Other sensory-stimulating alarm systems may be used includingalarm systems which heat, cool, vibrate, or produce a mild electricalshock when activated.

Drug Delivery System

The subject invention also includes sensors used in sensor-based drugdelivery systems. The system may provide a drug to counteract the highor low level of the analyte in response to the signals from one or moresensors. Alternatively, the system may monitor the drug concentration toensure that the drug remains within a desired therapeutic range. Thedrug delivery system may include one or more (e.g., two or more)sensors, a processing unit such as a transmitter, a receiver/displayunit, and a drug administration system. In some cases, some or allcomponents may be integrated in a single unit. A sensor-based drugdelivery system may use data from the one or more sensors to providenecessary input for a control algorithm/mechanism to adjust theadministration of drugs, e.g., automatically or semi-automatically. Asan example, a glucose sensor may be used to control and adjust theadministration of insulin from an external or implanted insulin pump.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments of the invention, and are not intended tolimit the scope of what the inventors regard as their invention nor arethey intended to represent that the experiments below are all or theonly experiments performed. Efforts have been made to ensure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Cationic Polymer Based Sensing Layer Formulation Using GlucoseOxidase

Sensing layers were prepared that included either a control WiredEnzyme™ sensing layer system that includes redox polymer in which themediator is immobilized to the polymer, crosslinker, and glucose oxidaseor a cationic polymer based sensing layer (Table 1). Beaker calibrationand beaker stability experiments were performed to compare the cationicpolymer based sensing layer to the control sensing layer.

TABLE 1 Cationic Polymer Based GOx Sensing Layer Formulation mL HepesBuffer, 10 mM, pH 8 0.09 Polyllyamine Hydrochloride (MW 60K), 10 mg/mL0.03 OsPLX, 10 mg/mL 0.03 GOX from Toyobo, 134 U/mg, 10 mg/mL 0.05 Total0.2

FIG. 6 shows that the cationic polymer based sensing layer (triangle)maintains a linear sensitivity to increasing concentrations of glucosesimilar to that of the control sensing layer (diamond). FIG. 7 showsthat the cationic polymer based sensing layer has a response time toglucose that is at least similar to and in some instances shorter thanthat of the control sensing layer.

FIG. 8 and FIG. 9 show beaker stability in 30 mM glucose at either 37°C. (FIG. 8) or 65° C. The results show that the cationic polymer basedsensing layer is much more stable than the control sensing layer and hasa lower rate of decay as shown in Table 2.

TABLE 2 Different GOx Sensing Layer Stability at 65° C. Sensing LayerControl Cationic Polymer Based Sensing Layer Sensing Layer Decay rate(per hour) 3.97% 1.67%

In summary, as compared to a control sensing layer that includes a redoxpolymer and crosslinker, the cationic polymer based GOx sensing layersprovide a linear response to varying concentrations of glucose, have ashorter response time, and are more stable and exhibit a lower rate ofdecay.

Example 2 Cationic Polymer Based Sensing Layer Formulation Using GlucoseDehydrogenase

Sensing layers were prepared that included either a control WiredEnzyme™ sensing layer system or the cationic polymer sensing layer(Table 3). The control system included redox polymer, crosslinker, andFADGDH. Beaker calibration and beaker stability experiments wereperformed to compare the cationic polymer based sensing layer to thecontrol sensing layer.

TABLE 3 Cationic Polymer Based FAD Sensing Layer Formulation Final Con-Volume centration mL mg/mL Hepes Buffer, 10 mM pH 8 0.016 PolyallyamineHydrochloride (MW 60K) (PAH), 0.03 3.0 20 mg/mL FADGDH from Toyobo,7507A, 540 U/mg, 0.12 12.0 20 mg/mL OsPLX, 10 mg/mL 0.034 1.7 Total 0.2

FIG. 10 shows that the cationic polymer based sensing layer (square)maintains a linear sensitivity to increasing concentrations of glucosesimilar to that of the control sensing layer (diamond). FIG. 11 showsthat the cationic polymer based sensing layer has a response time toglucose that is shorter than that of the control redox polymer andcrosslinker based sensing layer.

FIG. 11 shows beaker stability in 15 mM glucose for the control redoxpolymer and crosslinker based sensing layer and in 20 mM glucose for thecationic polymer based sensing layer at 65° C. The results show that thecationic polymer based sensing layer is much more stable than thecontrol redox polymer and crosslinker based sensing layer and has alower rate of dacay as shown in Table 4.

TABLE 4 Different FADGDG Sensing Layer Response Time and StabilitySensing Layer Control Cationic Polymer Based Sensing Layer Sensing LayerResponse Time (h:mm:ss) 0:06:30 Decay rate (per hour) 3.97% 1.67%

In summary, as compared to a control sensing layer that includes redoxpolymer and crosslinker, the cationic polymer based FADGDH sensing layerprovides a linear response to varying concentrations of glucose, have ashorter response time, and are more stable and exhibit a lower rate ofdecay.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the embodiments ofthe invention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe embodiments of the invention are exemplified by the appended claims.

1-60. (canceled)
 61. An analyte sensor assembly, comprising: anelectrochemical sensor comprising: a substrate comprising a workingelectrode and a counter electrode or a counter/reference electrode; asensing layer disposed on the working electrode, wherein the sensinglayer comprises a glucose-responsive enzyme, a cationic polymer selectedfrom the group consisting of polyallylamine (PAH), polyethyleneimine(PEI), poly(L-lysine) (PLL), or poly(L-arginine) (PLA), and a positivelycharged redox mediator non-covalently associated with the cationicpolymer; and a transmitter unit operatively coupled to theelectrochemical sensor to receive signals from the electrochemicalsensor corresponding to an analyte level of a subject.
 62. The analytesensor of claim 61, wherein at least a portion of the sensor is adaptedto be subcutaneously positioned in a subject.
 63. The analyte sensor ofclaim 61, wherein the analyte-responsive enzyme is glucose oxidase(GOx).
 64. The analyte sensor of claim 61, wherein theanalyte-responsive enzyme is a dehydrogenase.
 65. The analyte sensor ofclaim 64, wherein the dehydrogenase is glucose dehydrogenase (GDH). 66.The analyte sensor of claim 65, wherein the glucose dehydrogenase isassociated with a co-factor.
 67. The analyte sensor of claim 66, whereinthe co-factor is flavin adenine dinucleotide (FAD), nicotinamide adeninedinucleotide (NAD), or pyrroloquinoline quinone (PQQ).
 68. The analytesensor of claim 64, wherein the dehydrogenase comprises a complex ofglucose dehydrogenase (GDH) and flavin adenine dinucleotide (FAD). 69.The analyte sensor of claim 61, further comprising a flux limiting layerdisposed over at least a portion of the working electrode.
 70. Theanalyte sensor of claim 61, wherein the redox mediator comprisesferricyanide, phenanthroline quinone, or ferrocene.
 71. The analytesensor of claim 61, wherein the redox mediator comprises a transitionmetal complex.
 72. The analyte sensor of claim 71, wherein thetransition metal complex comprises osmium.
 73. The analyte sensor ofclaim 61, wherein the transition metal complex comprises the followingformula:

wherein (i) M is ruthenium, osmium, or vanadium; and (ii) L is selectedfrom the group consisting of:

wherein: R₁, R₂, and R′₁ are independently substituted or unsubstitutedalkyl, alkenyl, or aryl groups; R₃, R₄, R₅, R₆, R′₃, R′₄, R_(a), R_(b),R_(c), and R_(d) are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, —NHNH₂, —SH, aryl, alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, —OH, alkoxy, —NH₂, alkylamino, dialkylamino,alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl; c is aninteger selected from −1 to −5 or +1 to +5 indicating a positive ornegative charge; X represents at least one counter ion; d is an integerfrom 1 to 5 representing the number of counter ions; and L₁, L₂, L₃ andL₄ are ligands, wherein L₁ comprises a heterocyclic compound coupled apolymeric backbone; and wherein L₁ and L₂ in combination form a firstbidentate ligand.
 74. The analyte sensor of claim 73, wherein thetransition metal complex comprises the following formula:

wherein R₃, R₄, R₅, R₆, R_(a), R_(b), R_(c), R_(d), R′₃ and R′₄ are —H;R₁ and R₂ are independently substituted or unsubstituted C1 to C12alkyls; and R₁, R₂, and R′₁ are independently —H or substituted orunsubstituted C1-C12 alkoxy, C1-12 alkylthio, C1-C12 alkylamino, C2-C24dialkylamino, or C1-C12 alkyl.
 75. The analyte sensor of claim 74,wherein at least one of R₁, R₂, and R′₁ comprises a reactive groupselected from the group consisting of carboxy, activated ester, sulfonylhalide, sulfonate ester, isocyanate, isothiocyanate, epoxide, aziridine,halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acylhalide, hydrazine, hydroxyamine, alkyl halide, imidazole, pyridine,phenol, alkyl sulfonate, halotriazine, imido ester, maleimide,hydrazide, hydroxy, and photo-reactive azido aryl groups.